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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190096	1/2	Bos taurus	skim milk	acetic acid treatment DG (d)(U)C Filtration	Mukhopadhya, Anindya	2021	100%
Study summary Full title Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk All authors Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll Journal Food Chem. Abstract Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type skim milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture acetic acid treatment DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9 non-EV: Actinin4 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type skim milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 2.33 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 186000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: duration (min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.45µm > x > 0.22µm, Other Name other separation method acetic acid treatment Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101 Not detected EV-associated proteins Detected contaminants Not detected contaminants Actinin4 Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Flow Cytometer Calibration bead size none Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLADR Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 160 EV concentration Yes

	EV190096	2/2	Bos taurus	powdered infant milk formula	acetic acid treatment DG (d)(U)C Filtration	Mukhopadhya, Anindya	2021	100%
Study summary Full title Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk All authors Anindya Mukhopadhya, Jessie Santoro, Barry Moran, Zivile Useckaite, Lorraine O'Driscoll Journal Food Chem. Abstract Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves (show more...)Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM. (hide) EV-METRIC 100% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type powdered infant milk formula Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture acetic acid treatment DG (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD81/ HLADR/ ADAM10/ CD9 non-EV: Actinin4 Proteomics no EV density (g/ml) 1.15 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Bos taurus Sample Type powdered infant milk formula Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 17 Sample volume (mL) 2.33 Orientation Bottom-up Rotor type Type 70.1Ti Speed (g) 186000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 9 Pelleting: duration (min) 90 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.45µm > x > 0.22µm, Other Name other separation method acetic acid treatment Other Name other separation method Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ TSG101 Not detected EV-associated proteins Detected contaminants Not detected contaminants Actinin4 Flow cytometry Type of Flow cytometry AMNIS ImageStreamX Mark II Flow Cytometer Calibration bead size none Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD81/ ADAM10/ HLADR Not detected EV-associated proteins Detected contaminants Not detected contaminants Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 175 EV concentration Yes

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EV-TRACK ID	EV190096
species	Bos taurus
sample type	skim milk	powdered infant milk formula
condition	Control condition	Control condition
separation protocol	acetic acid treatment DG (d)(U)C Filtration	acetic acid treatment DG (d)(U)C Filtration
Exp. nr.	1	2
EV-METRIC %	100	100